This study examined the biochemical and histological changes associated with methanolic leaf extract of Gongronema latifolium in acetaminophen-induced hepatic toxicity in wistar albino rats. The serum liver enzymesALT, AST and ALP decreased significantly (p<0.05) in the test animals treated with 600mg/kg of the leaf extract. Protein concentration increased significantly (p<0.05) in the test animals treated with 600mg/kg of the leaf extract.The effect of the G. latifolium leaf extract seems to be dose dependent on the liver enzymes and protein concentration measured. The results showed that acetaminophen-induced hepatic toxicity in the wistar albino rats as observed in the negative control was reversed with the administration of the leaf extract of Gongronema latifolium (in groups 3, 4 and 5) in the test animals. The histological analysis of the liver showed that the extract had a normalising effect on the effected liver. These results indicate that the leaf extract of Gongronema latifolium exhibits biochemical and histological changes and can be used against some hepatic inflammations.

1. 1 Int. J. Biomol. Biomed.
International Journal of Biomolecules and Biomedicine (IJBB)
ISSN: 2221-1063 (Print) 2222-503X (Online)
Vol. 4, No. 2, p. 1-7, 2014
http://www.innspub.net
RESEARCH PAPER OPEN ACCESS
Biochemical and histological changes associated with methanolic leaf
extract of Gongronema latifolium in acetaminophen-induced hepatic
toxicity in wistar albino rats
Chinedu Imo1*
, Friday O. Uhegbu1
, Ifeanacho Nkeiruka G.2
, Egbeigwe Otito1
and Ezekwe
A. S.3
1
Department of Biochemistry, Abia State University, P. M. B. 2000, Uturu, Abia State, Nigeria
2
Department of Animal Production and Management, Michael Okpara University of Agriculture,
Umudike, Abia State, Nigeria
3
Department of Medical Biochemistry, Faculty of Medicine, Imo State University, Owerri, Imo State,
Nigeria
Article Published: 03 June 2014
Key words: Acetaminophen, biochemical, Gongronema latifolium, histological changes.
Abstract
This study examined the biochemical and histological changes associated with methanolic leaf extract of
Gongronema latifolium in acetaminophen-induced hepatic toxicity in wistar albino rats. The serum liver enzymes
ALT, AST and ALP decreased significantly (p<0.05) in the test animals treated with 600mg/kg of the leaf extract.
Protein concentration increased significantly (p<0.05) in the test animals treated with 600mg/kg of the leaf
extract.The effect of the G. latifolium leaf extract seems to be dose dependent on the liver enzymes and protein
concentration measured. The results showed that acetaminophen-induced hepatic toxicity in the wistar albino rats as
observed in the negative control was reversed with the administration of the leaf extract of Gongronema latifolium
(in groups 3, 4 and 5) in the test animals. The histological analysis of the liver showed that the extract had a
normalising effect on the effected liver. These results indicate that the leaf extract of Gongronema latifolium exhibits
biochemical and histological changes and can be used against some hepatic inflammations.
*Corresponding Author: Chinedu Imo  chinedu04@yahoo.com
Imo et al.

2. 2 Int. J. Biomol. Biomed.
Introduction
Gongronema latifolium (whose leaves are bitter) is
commonly called ‘‘utazi’’ and ‘‘arokeke’’ in South
Eastern and South Western parts of Nigeria
respectively. It is a tropical rainforest plant primarily
used as spice and vegetable in traditional folk medicine
(Ugochukwu et al., 2003).
Phytochemical analysis of leaf extract of Gongronema
latifolium shows the presence of essential oil,
saponins, alkaloids, minerals with calcium,
phosphorus, magnesium, copper and potassium
(Atangwho et al., 2009). It is a tropical rainforest plant
which has been traditionally used in the South Eastern
part of Nigeria for the management of diseases such as
diabetes and high blood pressure. The presence of
phytochemicals (tannins, saponins, alkaloids,
flavonoids and hydrocyanide), proximate (crude fat,
ash, fat and protein), mineral elements (Cr, Cu, Se, Zn
and Fe) and vitamins (A, C, riboflavin, niacin and
thiamine) has been reported in the root bark and twig
extracts (Egbung et al., 2011). Plant based natural
constituents can be derived from any part of the plant
like bark, leaves, flowers, roots, fruits, seeds, etc. that is
any part of the plant may contain active components
(Ibe et al., 2014).
Elevated levels of serum enzymes are inductive of
cellular leakage and loss of functional integrity of cell
membrane in liver. Since the plant is used variously by
traditional/alternative medicine practitioners to
manage various ailments, the biochemical and
histological changes was therefore studied.
Acetaminophen (Paracetamol) is a widely used over-
the-counter analgesic and antipyretic. It is commonly
used for the relief of headaches and other minor aches
and pains and is a major ingredient in numerous cold
and flu remedies (Chinedu et al., 2013). The onset of
analgesia is approximately 11 minutes after oral
administration of paracetamol (Moller et al., 2005),
and its half-life is 1–4 hours. Though acetaminophen is
used to treat inflammatory pain, it is not generally
classified as an NSAID because it exhibits only weak
anti-inflammatory activity.
While generally safe for use at recommended doses
(1,000 mg per single dose and up to 4,000 mg per day
for adults), acute overdose of paracetamol can cause
potentially fatal liver damage and, in rare individuals, a
normal dose can do the same; the risk is heightened by
alcohol consumption. Paracetamol toxicity is the
foremost cause of acute liver failure in the Western
world, and accounts for most drug overdose in the
United States, the United Kingdom, Australia and New
Zealand.
Histology studies the microscopic anatomy of cells and
tissues of plants and animals. It is commonly
performed by examining cells and tissues by sectioning
and staining, followed by examination under a light
microscope or electron microscope. The ability to
visualize or differentially identify microscopic
structures is frequently enhanced through the use of
histological stains. Histological assessment of the liver,
and thus, liver biopsy, is a cornerstone in the
evaluation and management of patients with liver
disease and has long been considered to be an integral
component of the clinician’s diagnostic
armamentarium (Rockey et al., 2009).
Liver histology may also be very helpful in patients
with coexisting disorders such as steatosis and HCV or
hemochromatosis or an “overlap” syndrome of PBC
with AIH (Zein et al., 2003).
Materials and methods
Plant material
The leaf of Gongronema latifolium was harvested at
Itaja-Amaegbu Olokoro in Umuahia, Abia State,
Nigeria.
Imo et al.

3. 3 Int. J. Biomol. Biomed.
The plant was identified at the Department of Plant
Science and Biotechnology, Abia State University,
Uturu and voucher specimen deposited at the
herbarium of the department. The plant material was
sun-dried. The dried leaf of Gongronema latifolium
was milled to a powder. About 250 gm of the powder
was extracted with 625 ml of methanol by cold
maceration for 48 hours and filtered. The filtrate was
evaporated to dryness using a soxhlet extractor and the
concentration of the extract determined.
Experimental design
Thirty male albino rats aged 8 weeks and weighing
between 120g-130g were used in this study. The
animals were randomly placed into five (5) groups of
six (6) rats in each group. Group 1 served as the control
group and received a placebo of 0.9% normal saline.
Group 2 were treated with acetaminophen (1000mg/kg
body weight) only and served as negative control.
Groups 3, 4 and 5 received concurrently
acetaminophen 1000mg/kg plus 200 mg/kg ,
400mg/kg, and 600mg/kg of G. latifolium leaf extract
respectively. The drug and extract was administered by
oral intubation. The treatment lasted for twenty-one
days. All animals were allowed free access to food and
water ad libitum throughout the study. Enas, 2012
reported that 1000mg/kg body weight of rats causes
liver damage.
All processes involved in the handling of animals and
the experiment was carried out according to standard
protocols approved by the animal ethics committee of
the College of Medicine and Health Sciences, Abia
State University, Uturu
Blood and Liver collection
Forty eight hours after treatment with the leaf extract,
the animals were starved overnight, anaesthetized with
chloroform and sacrificed. Blood was collected by
cardiac puncture and blood samples from each animal
collected into dry test tubes. The blood sample was
allowed to stand for about 15 minutes to clot and
further spun in a centrifuge. Serum was separated from
the clot with Pasteur pipette into sterile sample test
tubes for the measurement of liver enzymes and
protein concentration.
After sacrificing the animals, the liver of a
representative of each of the five groups were taken to
University of Nigeria Teaching Hospital Enugu for
histological analysis.
Biochemical analysis
The serum aspartate transaminase (AST) and alanine
transaminase (ALT) were determined as described by
Reitman and Frankel, 1956 using Randox Diagnostic
kit. Alkaline phosphatase (ALP) was determined as
described by Tietz et al., 1983 also using Randox
Diagnostic kits. The assays were performed according
to the manufacturer’s instructions. Serum total protein
was determined using Biuret method as described by
Henry et al., 1974.
Statistical analysis
Data were expressed as mean ± standard deviation.
The statistical evaluations of the data were carried out
with the use of standard student T distribution test and
mean was compared for significant at (p≤0.05).
Results
The results of the study are presented in the table and
figures below.
Table 1: Protein concentration and liver enzymes concentration
Parameters Group 1 Group 2 Group 3 Group 4 Group 5
Protein (g/dl) 8.43 ± 0.42 3.68 ± 0.24 d 6.89 ± 0.25 c 7.29 ± 0.30 b 8.33 ± 0.26 a
ALT (U/L) 27.38 ± 3.80 35.38 ± 1.21a 32.97 ± 2.50 a 30.77 ± 1.62 b 28.35 ± 1.56 c
AST (U/L) 34.32 ± 1.97 45.98 ± 3.37 a 38.42 ± 0.96 b 36.43 ± 1.34 c 35.75 ± 1.94 c
ALP (U/L) 65.17 ± 1.29 84.33 ± 2.03 a 77.43 ± 1.90 b 71.70 ± 1.70 c 65.92 ± 3.49 d
* Results represent mean ± standard deviation of group serum results obtained (n=6).
Values in the same row with different superscript are statistically significant.
Imo et al.

4. 4 Int. J. Biomol. Biomed.
Fig. 1. Light photomicrograph of liver sections from Control rat (group 1).
Fig. 2. Light photomicrograph of sections from liver of rat administered with acetaminophen only (group 2).
Fig. 3. Light photomicrograph of sections from liver of rat administered with acetaminophen and G. latifolium leaf
extract [200mg/kg b.wt.] (group 3).
Imo et al.

5. 5 Int. J. Biomol. Biomed.
Fig. 4. Light photomicrograph of sections from liver of rat administered with acetaminophen and G. latifolium Leaf
extract [400mg/kg b.wt.] (group 4).
Fig. 5. Light photomicrograph of sections from liver of rat administered with acetaminophen and G. latifolium Leaf
extract [600mg/kg b.wt.] (group 5).
Discussion
Acetaminophen administration caused increase in liver
enzymes in the group 2 animals (negative control).
Hepatotoxic drugs cause damage to the liver. Elevated
levels of serum enzymes are inductive of cellular leakage
and loss of functional integrity of cell membrane in liver.
Concurrent administration of acetaminophen and
methanolic leaf extract of Gongronema latifolium as
seen in group 3, 4 and 5 reduced these elevated levels of
the liver enzymes. This decrease in serum liver enzymes
shows that the leaf extract could have ameliorating
properties. Researchers have reported that Gongronema
latifolium not only possess hypotensive and
hypolipidemic activity but also hepatoprotective activity.
Protein concentration reduced in the group 2, but
increased (p≤0.05) significantly in all the groups treated
with acetaminophen and methanolic leaf extract of
Gongronema latifolium (groups 3, 4 and 5). Most
protein found in the plasma are synthesized by the
hepatocytes and secreted into circulation. A reduction in
Imo et al.

6. 6 Int. J. Biomol. Biomed.
the protein levels in the serum (group 2) and hepatic
tissue may be a result of possible damage to the
hepatocytes induced by the ingested toxin. The serum
protein level is a marker of the synthetic function of the
liver and a valuable guide to assess the severity of the
damage (Nair, 2006).
The histological analysis of the liver of rat in the five
different groups show: normal histoarchitecture of
pericentral and periportal regions of the hepatic tissue
(the central vein, hepatocytes interacting with the
sinusoidal spaces and portal tract are evident) in group
1 (Fig. 1), distorted histoarchitecture of periportal and
pericentral regions of the hepatic tissue (ballooning
degeneration of hepatocytes at pericentral region
shown; Increased septal and portal fibrosis, dilated
vessels, necrosis of periportal hepatocytes and
inflammatory cellular infiltration are also observed) in
group 2 (Fig. 2), slightly preserved histoarchitecture of
periportal and pericentral regions of the hepatic tissue
(inflammatory cellular infiltration is shown at
periportal regions; binucleates indicating regeneration
is also observed, presence of leucocytes and lysed red
blood cells are found within the central vein) in group 3
(Fig. 3), slightly preserved histoarchitecture of
periportal and pericentral regions (portal fibrosis,
vacuolation and lysed red blood cells within the central
vein are shown, presence of inflammatory cellular
infiltration at pericentral and periportal regions is
evident) in group 4 (Fig. 4) and some degree of
protection of the periportal and pericentral regions
(mild cellular infiltration and hepatic vacuolation are
observed at periportal regions; binucleates indicating
regeneration and anisonucleosis are also seen) in group
5 (Fig. 5).
Overdose of acetaminophen causes a potentially fatal,
hepatic necrosis which can be attributed to the
formation of a toxic metabolite N-acetyl-p-
benzoquinoneimine (NAPQI) by the action of cyt P450.
The reduced necrosis of cells in the group 3, 4 and 5
(treated with G. latifolium leaf extract) might be due to
the presence of chemical constituents which have
hepatoproctective properties. Liver protective herbal
drugs contain a variety of chemical constituents like
phenols, coumarins, lignans, essential oil,
monoterpenes, carotinoids, glycosides, flavonoids,
organic acids, lipids, alkaloids and xanthenes (Gupta
and Misra, 2006), this may be present in the
Gongronema latifolium and so responsible for this
changes.
From this study, methanolic leaf extract of
Gongronema latifolium has an appreciable ability to
prevent damage to the liver based on the biochemical
and histological changes observed.
Conclusion
The results of this study demonstrate that methanolic
leaf extract of Gongronema latifolium methanolic
exhibits biochemical and histological changes against
acetaminophen-induced hepatic damage in wistar
albino rats. It is possible that, the mechanism of the
biochemical and histological changes of Gongronema
latifolium methanolic leaf extract may be due to the
presence of chemical constituents which have
hepatoproctective, antioxidant and free radical
scavenging properties. Additionally, this is a clear
indication of the efficacy of methanolic leaf extract of
Gongronema latifolium as an antidote to human
health. It is pertinent, therefore to note that if adequate
attention is given to the findings of this study, the
Nigerian population and the world at large will
experience laudable progress.
References
Atangwho IJ, Ebong PE, Eyong EU, Williams IO,
Eteng MU, Egbung GE. 2009. Comparative chemical
composition of leaves of some antidiabetic medicinal
plants: Azadirachta indica, Vernonia amygdalina and
Gongronema latifolium. African Journal of
Biotechnology 8 (18), 4685-4689.
Imo et al.

7. 7 Int. J. Biomol. Biomed.
Chinedu I, Uhegbu FO, Imo CK, Ifeanacho NG.
2013. Ameliorating effect and haematological activities
of methanolic leaf extract of Gongronema latifolium in
acetaminophen-induced hepatic toxicity in wistar
albino rats. International Journal of Biosciences, 3 (11),
183-188.
Egbung GE, Atangwho IJ, Iwara IA, Eyong UE.
2011. Micronutrient and phytochemical composition of
root bark and twig extracts of Gongronema latifolium.
Journal of Medicine and Medical Sciences 2 (11), 1185-
1188.
Enas AKM. 2012. Hepatoprotective effect of aqueous
leaves extract of Psidium guajava and Zizyphus spina –
christi against paracetamol induced hepatotoxicity in
rats. Journal of Applied Sciences Research 8 (5), 2800
- 2806.
Gupta AK, Misra N. 2006. Hepatoprotective Activity
of Aqueous Ethanolic Extract of Chamomile capitula in
paracetamol intoxicated albino rats. American Journal
of Pharmacology and Toxicollogy 1, 17-20.
Henry RJ, Cannon DC, Winkelman JW. 1974.
Clinical chemistry, principles and techniques. Harper
and Row 2nd Edition, 943-949.
Ibe C, Conrad CJ, Chinedu I, Kelechi UO,
Monday UO. 2014. Evaluation of the Antioxidant
Activities of Psidium guajava and Aloe vera. British
Journal of Pharmaceutical Research 4(3), 397-406.
Moller P, Sindet-Pedersen S, Petersen C, Juhl G,
Dillenschneider A, Skoglund L. 2005. Onset of
acetaminophen analgesia: comparison of oral and
intravenous routes after third molar surgery. British
Journal of Anaesthesia 94 (5), 642–648.
Nair SP. 2006. Protective effect of Tefroli- a
polyherbal mixture (tonic) on cadmium chloride
induced hepatotoxic rats. Pharmacognosy Magazine 2
(6), 112-118.
Reitman S, Frankel S. 1956. Transaminases.
American Journal of Clinical Pathology 28, 56.
Rockey DC, Caldwell SH, Goodman ZD, Nelson
RC, Smith AD. 2009. Liver Biopsy. Hepatology 49
(3), 1017.
Tietz NW, Burtis CA, Duncan P, Ervin K,
Petitclerc CJ, Rinker AD, Shuey D, Zygowicz ER.
1983. A reference method for measurement of alkaline
phosphatase activity in human serum. Clinical
Chemistry 3 (29), 751-761.
Ugochukwu NH, Babady NE, Cobourne M,
Gasset SR. 2003. The effect of Gongronema latifolium
extracts on serum lipid profile and oxidative stress in
hepatocytes of diabetic rats. Journal of Biosciences 20
(1), 1-5.
Zein CO, Angulo P, Lindor KD. 2003. When is liver
biopsy needed in the diagnosis of primary biliary
cirrhosis? Clinical Gastroenterology and Hepatology 1,
89-95.
Imo et al.